The partial section of mitochondrial cytochrome oxidase Ⅰ(COⅠ) was sequenced. These sequences were in 52 individuals from four Bactrocera dorsalis geographic populations (Liuku, Dali, Kunming, Qujing) located in the seasonal occurrence zone of the fly in Yunnan Province. The 503bp sequences were obtained from mitochondrial cytochrome oxidaseⅠDNA sequences. Fifteen nucleotide sites were substituted. Fourteen haplotypes were identified in all the sequences, with four shared. The genetic distances of the four populations were analyzed by K2P genetic distances and Fst value. Average number of pairwise genetic differences were obtained between and within 4 populations. Gene flow among four populations was also detected. All results showed that genetic differences existed among the four populations. The genetic differences between Liuku and other three populations were largest. The p value was <0.05. There were some genetic differences between Dali and Qujing population. That p value <0.01. But the genetic differences between Kunming and Qujing population were lowest. In a word, in seasonal occurrence zone of Bactrocera dorsalis , the genetic differences was larger in the western region. The genetic differences declined gradually from western to eastern region. The two populations located in eastern region have closer genetic structure. The results showed geographic isolation is the main reason of genetic differences between Liuku population and other three populations. The lower genetic differences among Dali, Kunming and Qujing population probably resulted from them having the same origin. The results of the study have important implications for the management and eventual elimination of Bactrocera dorsalis populations at different locations in the seasonal occurrence zone in Yunnan.